Liquid chromatography (LC), e.g. HPLC and UHPLC, and solid phase extraction (SPE) are used routinely in both analytical and preparative chromatography applications. In these chromatographic techniques, separation of a sample comprising a mixture of components (also termed analytes) is achieved by conveying the sample in a liquid mobile phase through a stationary phase in a column, thereby causing the sample to separate into its components due to different partitioning between the mobile and stationary phases of each of the components (i.e. the components have different partition coefficients). The stationary phase is typically in the form of a bed of particles packed within the column, or in the form of a monolithic material held in the column.
A bed of non-porous particles has a relatively low sample capacity. Therefore, porous particles are commonly used which contain a network of pores to increase the surface area of the stationary phase and thus improve the capacity of the separation. The porous particles may be fully porous, wherein the pores extend throughout the bulk of the particles. As an alternative to fully porous particles, more recently use has been made of so-called fused core particles, which are also termed superficially porous particles. These are particles that have a non-porous core (also termed a fused or solid core) and are porous only in an outer layer or region that surrounds the non-porous core. Silica particles are commonly used as the stationary phase, either as non-porous, fully porous or superficially porous particles.
The selectivity of a stationary phase for analytes is mainly governed by column chemistry, which is key in LC separation. Although reversed-phase (RP) columns (e.g. columns comprising a C18 stationary phase) are most commonly used in pharmaceutical applications, they often fail to retain highly polar molecules (e.g. counter ions) and offer limited selectivities. Instead, ion exchange (IEX) chromatography is typically used to separate ionic or ionizable molecules. However, IEX has limited use in organic molecule separations due to inadequate hydrophobic retention. Ion Pairing chromatography can help to alleviate the aforementioned difficulties by allowing ionic analytes to be separated on a reversed-phase column but it has drawbacks in that it often requires extended equilibration time, a complicated mobile phase with high salt content that is incompatible with mass spectrometry (MS), and a dedicated column.
Mixed-mode chromatography provides a viable solution to the aforementioned challenges by utilizing a stationary phase that provides both reversed-phase and ion-exchange retention mechanisms. An advantage of the mixed-mode approach is that column selectivity can easily be modified by adjusting mobile phase ionic strength, pH and/or organic solvent concentration. As a result, not only is the selectivity of a mixed-mode column complementary to that of reversed-phase columns, but it also allows for the development of multiple complementary selectivities on a given column under different appropriate conditions. Mixed-mode chromatography is well-suited to retaining ionic, hydrophobic (e.g. Naproxen), or hydrophilic (e.g. Na+ and Cl− ions) analytes, and requires no ion-pairing agents, thereby significantly improving compatibility with MS. Many applications involving hydrophilic ionizable compounds that are problematic on a C18 column are easily addressed on a mixed-mode column. The use of the mixed-mode technique has been growing rapidly because of its advantages over conventional chromatography, such as its high resolution, adjustable selectivity, high sample loading, and its lack of need for ion-pairing agents.
Mixed-mode media can be classified into at least four categories based on column chemistry. The first type includes a blend of two different stationary phases (RP and IEX) (such as Thermo Scientific HYPERSIL™ Duet C18/SCX Mixed-mode Ion Exchange columns). The second type involves bonded silica modified by a mixture of both RP and IEX ligands in the bonding step (such as ALLTECH™ Mixed-Mode Columns). EP 2745903 A1 (Dionex) discloses the use of two or more different chromatographic moieties bound to a solid support, which have anion-exchange capabilities, cation-exchange capabilities, reverse-phase capabilities, or hydrophilic interaction capabilities. Although these first two types of media are relatively straightforward to synthesize, their use in many applications is limited by selectivity drifting, mainly due to the difference in hydrolytic stability between the RP and IEX ligand bonded sites.
The third and fourth types of mixed-mode media are more recent and use functional silyl ligands that contain both RP and IEX functionalities to covalently attach to silica particles. While the constant ratio between RP and IEX bonded sites greatly improves the selectivity robustness in these materials, a pronounced distinction exists between the third and fourth types. The third type of material uses IEX-embedded alkyl silyl ligands to modify the silica and can be viewed as an IEX-modified RP packing (such as PRIMESEP™ and OBELISC™ columns from SIELC Technologies). Further disclosure is contained in US 2005-0023203 A and in US 2006-0169638 A. By comparison, the fourth type material uses IEX-tipped silane ligand to functionalize the silica substrate, (such as disclosed in U.S. Pat. No. 7,402,243 and embodied in ACCLAIM™ Mixed-Mode columns from Thermo Fisher Scientific).
A technique that utilises a hydrophobic chain in combination with a zwitterionic end group is known as Immobilized Artificial Membrane Chromatography (IAMC). WO 89/08130, WO 91/11241 and D. E. Cohen and M. R. Leonard, Journal of Lipid Research, Volume 36, 1995, p. 2251-2260 describe this application and associated chemical structures. Specifically the hydrophobic chain and zwitterionic portion aims to mimic a biological membrane and is designed for the separation of biomolecules.
US 2010/0300971 A discloses phosphorylcholine-type zwitterionic moieties in the absence of a reversed phase component that are bound to a solid support for hydrophilic interaction liquid chromatography (HILIC). A maximum distance between the negative charge and the solid support of 10 atoms is specified.
In Greco et al, J. Sep. Sci., 2013, 36, 8, 1379-1388, a technique is described wherein a reversed phase column is coupled with a zwitterionic phase column in series, with the obviously drawback of requiring two separate columns.
In L. W. Yu et al, Zwitterionic Stationary Phases in HPLC, Journal of Chromatographic Science, Vol. 27, April 1989, p. 176-185 is disclosed zwitterionic ligands, wherein relatively short ethyl or phenyl spacers are used as the linking group to the substrate, which limits their usefulness for reverse-phase chromatography.
In another approach, known as dynamically coating, a RP silica has been mixed with, for example, a zwitterionic surfactant having a hydrophobic tail group. The hydrophobic tail of the surfactant and the hydrophobic group bound to the silica associate together and form a co-mixture. Since the two components are not covalently attached to one another there is high potential to lose the zwitterionic surfactant from the system, thereby changing the chromatographic characteristics. A discussion of such phases and a general overview of zwitterionic phases can be found in E. P. Nesterenko et al, Analytica Chimica Acta 652 (2009) 3-21.
A recently developed class of mixed-mode separation media has been prepared by coating a porous solid support possessing a hydrophilic charged surface with polymer latex particles having the opposite charge via electrostatic attraction (see Journal of Chromatography A, 1218 (2011) 3407-3412 and ACCLAIM™ Trinity columns from Thermo Fisher Scientific). Due to the size of the latex particles (>50 nm) being larger than the size of the pores of the porous solid support (<30 nm), the outer surface of the support is functionalized by charged latex particles while the inner-pore area is intact and remains its original functionality and properties. Thus, the size-exclusion effect provides effective spatial separation between the inner-pore area and the outer surface so that the resulting material provides cation-exchange and anion-exchange properties at the same time. In addition, when the porous solid support is modified with hydrophilic interaction liquid chromatography (HILIC) and ion-exchange mixed-mode functionalities, after coating with the charged latex particles, the resulting material provides hydrophilic interaction liquid chromatography (HILIC), cation-exchange and anion-exchange functionalities (X. Liu and C. A. Pohl, J. Sep. Sci. 2010, 33, 779-786).